Transient analysis for antiproliferative gene activity.
A subset of tumor suppressor genes presumably functions by the inhibition of cellular proliferation; however, antiproliferative activity after transfection with putative suppressor genes has been difficult to demonstrate and often requires lengthy selection either in nude mice or in vitro. A rapid alternative is presented here that utilizes a gene encoding a surface marker protein to identify transfectants in a transient expression assay. In this assay the labeling index, rate of DNA synthesis, cell-cycle distribution, and surface receptor display are measured by flow cytometry. Human beta-interferon, a gene with proven antiproliferative activity, was studied using the transient analysis system. The beta-interferon gene was introduced into human tumor cells along with the marker gene encoding the 55-kDa subunit of the human interleukin 2 receptor. Within a few days of transfection, analysis of transfectants by flow cytometry revealed a decrease in the fraction of cells in G2/M and an increase in the fraction of cells in G1/G0 and S phases. The distortion of the cell cycle was accompanied by as much as a 69% reduction in the rate of DNA synthesis and, in some experiments, an unanticipated increase in the labeling index. Therefore, cells accumulating in S phase were not blocked but continued to synthesize DNA although at a reduced rate. These studies on DNA synthetic rates revealed the caveat that screening for antiproliferative candidate genes with a labeling index alone could, in certain circumstances, exclude potentially interesting sequences from further consideration. Although this transient analysis system was developed for studies on cellular proliferation, it may prove suitable for phenotypic assays on other genes as well.
Fordis
C M
CM
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Helmly
B
B
Novotny
E
E
Holter
W
W
Barker
J L
JL
Howard
B H
BH
D001973:Bromodeoxyuridine / Q000378:metabolism; D002453:Cell Cycle; D002455:Cell Division; D004261:DNA Replication; D005434:Flow Cytometry; D005796:Genes; D006367:HeLa Cells / Q000166:cytology; D006801:Humans; D007370:Interferon Type I / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D010957:Plasmids; D013489:Suppression, Genetic; D014162:Transfection
D007370:Interferon Type I; D008565:Membrane Proteins; D001973:Bromodeoxyuridine